The cholecystokinin (CCK) receptor is a family A peptide-binding G protein-coupled receptor that has multiple important physiologic functions supporting nutritional homeostasis, including the stimulation of pancreatic exocrine secretion and gallbladder emptying, the regulation of gastric emptying and bowel transit, and the modification of appetite. Like many other members of this superfamily, this receptor is a potentially important drug target, based on its role to induce satiety that can be useful in the prevention and/or treatment of obesity. The long-term goal of the current proposal is to enable more rational and directed CCK receptor drug development, built upon our recent observations that distinct small molecule CCK receptor ligands can have an allosteric mode of action and that the lipidic microenvironment of the type 1 CCK receptor can lead to dysregulation of receptor signaling. The proposed projects fall under two major aims. The first aim is receptor-centric, directed toward the elucidation of the structure of the small molecule-binding pocket within the helical bundle of the receptor and the chemistry of ligands binding there to achieve allosteric enhancement of the action of CCK. There are three sub-aims directed toward the following: (1.1) gaining detailed structural insights into the molecular basis of selectivity for the types 1 and 2 CCK receptors using mutant and chimeric receptors; (1.2) gaining insights into the determinants for biological activity of small molecules acting within this pocket using receptor mutagenesis; and (1.3) characterizing conformational changes in the cytosolic effector face of this receptor correlating with full agonist, partial agonist, and biased agonist activities using novel fluorescence techniques. The second aim is environment-centric, directed toward the lipid microenvironment of the CCK receptor and its impact on type-selective CCK stimulus-activity coupling. There are three sub-aims directed toward the following: (2.1) determining the structural basis for cholesterol sensitivity of the type 1 CCK receptor using mutant and chimeric receptors; (2.2) developing stable model cell lines having elevated membrane cholesterol and cell impermeant fluorescent probes of membrane cholesterol composition that can be utilized for morphological analysis, and that can be applied to flow analysis and sorting of living cells; and (2.3) using these reagents to more fully characterize the impact of an abnormal membrane lipid environment on type 1 CCK receptor function, and to develop and validate a strategy to normalize CCK stimulus-activity coupling in the setting of excess membrane cholesterol. Together, these activities should reenergize efforts to target the CCK receptor for the prevention and treatment of obesity, providing strategies for the development of novel allosteric enhancers that could be safer and more effective than existing full agonist candidate drugs, thereby exerting a sustained and powerful influence on a major public health problem.